| Antibodies are not widely used in the industrial and commercial application, especially in the field of analytical detection and theoretical research, which are mainly resulted from low-content of specific antibodies in the total antibodies with conventional extraction methods. In recent years, the isolation and purification of Immunoglobulin (IgY) have been concentrated on the combination and optimization of some chemical methods, and there is still no fixed and standard way to get specific IgY. Therefore, it is urgent to build a set of efficient and precise method to isolate specific IgY, which can provide reliable data and theoretical basis to develop immunoaffinity column. On the one hand, the situations of preparing immunoaffinity column have been optimized in various respects. On the other hand, crude IgY was isolated from layer chicken immunized by tyrosinase. Based on the research above and antigen-antibody reaction, immunoaffinity column was prepared to purify the specific IgY, with titer and enzymatic activity inhibition effects to determine the isolation effects.The main results were as follows:1、Coupling conditions of tyrosinase and column packing were set in normal design of three factors and three levels, which was determined on single factor experiment. The optimal coupling conditions and amount were, with coupling concentration of 20 mg/mL, pH 9, temperature 4℃, coupling time 12h, and coupling amount of 17.54 mg/mL respectively. In addition, the effects of loading conditions on the final targeted products were discussed under the use of immunoaffinity column. The optimal loading conditions were:loading buffer concentration of 0.5 mg/mL, flow velocity of 0.5 mL/min. The property of immunoaffinity:column capacity was 0.2620 mg/mL; elution rate of column on targeted products used for 3 and 5 times were 88.26% and 70% respectively; the elution rate of targeted products was up to 88.73% under 4℃ for 10 days, showing ideal effects and highly practicability.2^ The property of IgY, produced through response to immunize tyrosinase as the antigen in chicken, has been studied. It revealed that the content of specific IgY against tyrosinase was in stable and ideal efficacy level after three times of enhanced immune injection. On the seventh week, the titer of the specific IgY reached about 1:1000.The results of the physicochemical property indicated that IgY can remain immunological competence under different temperature and pH values. The bioactivity and efficacy of IgY were invariant with the temperature between 35-70℃; the efficacy of IgY remained above 50% with pH 4-7, showing great stability. Crude extraction of IgY in negative-control groups and positive-control groups showed some inhibitory ability in the enzymatic activity inhibition experiments of crude extraction of yolk antibody. The average enzymatic activity inhibition rate of positive control groups were above 27%, and negative control groups were about 16%. It could be concluded that crude extraction of yolk antibody could inhibit the enzymatic activity of polyphenol oxidase, but not significantly and stably.3、Specific IgY against tyrosinase could be extracted and purified through immunoaffinity column, with recovery rate of 62.91%. The immunoaffinity column has significant effects on isolation and purification of IgY specific against tyrosinase, the efficacy of which were 18 times higher than crude extraction of IgY. In the experiment of enzymatic activity inhibition, specific IgY exhibited efficient inhibition, with inhibition rate of up to 80%, which could come to the same conclusion when applied to different concentrations of enzyme. |
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